Homeostasis of the corneal epithelium is ultimately achieved by proliferation and differentiation of limbal epithelial stem cells (SC) located between the cornea and the conjunctiva. A number of human corneal diseases have now been found to exhibit limbal SC deficiency as a result of destruction of the limbal SC population or dysfunction of the limbal stroma. Patients with limbal deficiency suffer from severe visual loss and annoying ocular discomfort, and are poor candidates for conventional corneal transplantion. Transplantation of autologous limbal SC (limbal autograft) has been used to restore the normal corneal epithelium and vision. The major drawback of this surgical procedure is that the donor eye with the removal of one third to one half of the normal limbus may eventually develop limbal deficiency. The progress report shows that transplantation of amniotic membrane as a substrate helps restore a non-inflamed limbal stroma and expand the remaining limbal SC population. This result prompts the hypothesis that limbal SC population can be expanded on amniotic membrane cultures from a small limbal biopsy ( about1 mm3) using amniotic membrane cultures (Aim 1). This culture system will allow exploration of the pathogenesis of limbal deficiency. Specifically, they will examine the role of the basement membrane in supporting limbal SC expansion (Aim 2), and the pathogenic role of inflammation is causing limbal deficiency by analyzing how inflammatory cytokines may degrade the basement membrane by upregulating matrix metalloproteinases (MMPs) (Aim 3). Finally, they will verify the pre-clinical efficacy of this new approach in treating rabbit limbal deficiency in vivo (Aim 4). Completion of these aims will allow use of this new strategy of ex vivo expansion of limbal SC on amniotic membrane as a new surgical procedure to eliminate current problems of limbal autograft. Furthermore, the new knowledge thus generated will also help to develop a new therapy for preventing limbal deficiency, and pave the way for smilar manipulations of conjunctival SC for the purpose of conjunctival surface reconstruction in the future. Ex vivo expansion of epithelial SC is also a first step for gene therapy targeted at the SC level, and for tissue engineering of an epithelial tissue for future therapeutics and reconstruction.